Project Summary Alcohol use disorder (AUD) affects more than 16 million Americans, causing a substantial burden on those affected, the health care system, and the economy. Despite effective treatments, the majority of patients relapse and are unable to achieve long-term treatment success. Given the high rates of relapse, interventions that aim to prevent relapse have high potential impact. Research in both animal models and humans shows that early stages of sobriety are characterized by hyperarousal, anxiety, and depression. These symptoms are thought to result from the neuroadaptive changes that occur in response to chronic alcohol use and can persist after the initial detoxification into a stage known as prolonged withdrawal (PW). During PW, relapse is often driven by negative reinforcement, where drinking is reinforced by the removal of negative affect. Thus, PW is a high-risk period of recovery because it couples negative reinforcement with hyperarousal, anxiety, and high susceptibility to stress. In rodents, the bed nucleus of the stria terminalis (BNST) has been identified as a crucial brain region for both anxiety and stress-sensitivity, especially during prolonged withdrawal. New findings in an animal model of prolonged withdrawal suggest that insula projections to the BNST underlie the anxiety and depression phenotype typically observed during this withdrawal period. Our preliminary data show that the insula and BNST are also connected in humans. The insula is a complex region, with a heterogeneous structure, function, and connectivity. Findings to date show that the anterior insula is involved in emotion and reward while the posterior insula is involved in the perception of autonomic activity. Further, the anterior and posterior insula have distinct neurocircuits. The goal of the current project is to determine the extent to which BNST-insula connectivity is altered in humans during prolonged withdrawal. Three inter-related aims are proposed to achieve this goal. Specific Aim 1 will establish the normative pattern of BNST-insula structural connectivity (diffusion tensor imaging) and resting-state functional connectivity in healthy humans. An anterior and posterior division of the insula will be used to identify more precise patterns of BNST-insula connectivity. Specific Aims 2 and 3 will compare patients with AUD who are 30-180 abstinent (PW) to healthy controls (HC). Specific Aim 2 will test for alterations in BNST-insula structural and resting-state functional connectivity in the PW group compared to HC. Specific Aim 3 will investigate BNST-insula task-based functional connectivity during a mildly stressful task in the PW group compared to HC. The hypotheses are 1) that the anterior insula will have stronger structural and functional connectivity with the BNST than the posterior insula and 2) that BNST-anterior insula connectivity will be stronger in the PW group compared to HC. The results of this study will fill a critical knowledge gap, by elucidating the neural mechanisms involved in PW, which can be used to guide future targets for prevention and treatment of AUDs.